Emotional Stress Induces Adaptive Response in Rat Lymphocytes to Subsequent Ionizing Radiation Exposure.

We studied the molecular mechanisms of cross-adaptation to ionizing radiation (1 Gy) of lymphocytes isolated from rats subjected to emotional stress. The effects of chronic (CES; various types of stress exposure) and acute (AES; forced swimming) emotional stress in rats on indicators of oxidative stress, cell death, and levels of NRF2 and NOX4 proteins involved in the development of the adaptive response were analyzed in isolated lymphocytes. It was found that stress induced an adaptive response in rat lymphocytes and triggered processes similar to the adaptive response induced by low doses of ionizing radiation: an increase in the level of oxidized DNA and cell death, as well as an increase in the content of NOX4 and NRF2 proteins. In animals subjected to emotional stress, suppressed DNA oxidation in response to irradiation, reduced levels of protective factor NRF2, as well as lymphocyte death were observed.

[Antioxidant status of blood plasma of acutely psychotic patients and its correlation with Nrf2 activation]. / Antioksidantnyi status plazmy krovi patsientov s ostrym psikhozom i ego svyaz' s aktivatsiei transkriptsionnogo faktora Nrf2.

OBJECTIVE: To study the correlation between the blood plasma antioxidant profile and the transcriptional activity of the Nrf2 gene in acute psychosis in patients with schizophrenia and alcoholism. MATERIAL AND METHODS: The study included 40 patients with the first episode of the paranoid form of schizophrenia, 33 patients with schizophrenic psychosis who had previously received therapy, 22 patients with first-time acute alcohol psychosis, and 25 healthy volunteers. The level of Nrf2 in peripheral blood mononuclear cells was estimated by flow cytometry, and the antioxidant profile of blood plasma was estimated with chemiluminometry. RESULTS: The total and «thiol¼ antioxidant capacity were reduced in patients with initially diagnosed schizophrenic psychosis and alcoholic psychosis. In patients after treatment, the total antioxidant capacity was higher compared to previously untreated patients. The level of Nrf2 protein in mononuclear cells in patients with the first psychotic episode was significantly lower than in patients with alcoholism and lower than in the control group. In patients with alcoholic psychosis, Nrf2 level was correlated with both the total antioxidant capacity due to uric acid and the «thiol¼ antioxidant capacity; in patients with psychosis in schizophrenia, Nrf2 level was correlated only with the «thiol¼ antioxidant capacity. CONCLUSIONS: The correlation between the total and «thiol¼ antioxidant capacity and the level of Nrf2 in mononuclear cells of patients with alcohol delirium indicates the undamaged state of the regulation. The absence of a correlation between the total antioxidant capacity and the level of Nrf2 in patients with schizophrenia indicates a disturbance of the activation of the Nrf2 pathway due to, possibly, a part associated with the participation of uric acid.

Copy number variations of satellite III (1q12) and ribosomal repeats in health and schizophrenia.

OBJECTIVE: Earlier we studied the copy number variations (CNVs) of ribosomal repeat (rDNA) and the satellite III fragment (1q12) (f-SatIII) in the cells of schizophrenia patients (SZ) and healthy controls (HC). In the present study we pursued two main objectives: (1) to confirm the increased rDNA and decreased f-SatIII content in the genomes of enlarged SZ and HC samples and (2) to compare the rDNA and f-SatIII content in the same DNA samples of SZ and HC individuals. METHODS: We determined the rDNA CN and f-SatIII content in the genomes of leukocytes of 1770 subjects [HC (N = 814) and SZ (N = 956)]. Non-radioactive quantitative hybridization method (NQH) was applied for analysis of the various combinations of the two repeats sizes in SZ and HC groups. RESULTS: f-SatIII in human leukocytes (N = 1556) varies between 5.7 and 44.7 pg/ng DNA. RDNA CN varies between 200 and 896 (N = 1770). SZ group significantly differ from the HC group by lower f-SatIII content and by rDNA abundance. The f-SatIII and rDNA CN are not randomly combined in the genome. Higher rDNA CN values are associated with higher f-SatIII index values in SZ and HC. The f-SatIII variation interval in SZ group increases significantly in the subgroup with the high rDNA CN index values (>300 copies). CONCLUSION: Schizophrenia patients' genomes contain low number of f-SatIII copies corresponding with a large ribosomal repeats CN. A scheme is proposed to explain the low f-SatIII content in SZ group against the background of high rDNA CN.

Accumulation of Circulating Cell-Free CpG-Enriched Ribosomal DNA Fragments on the Background of High Endonuclease Activity of Blood Plasma in Schizophrenic Patients.

INTRODUCTION: Schizophrenia (SZ) increases the level of cell death, leading to an increase in the concentration of circulating cell-free DNA (cfDNA). Ribosomal DNA (rDNA) contains many unmethylated CpG motifs that stimulate TLR9-MyD88-NF-κB signaling and the synthesis of proinflammatory cytokines. The number of rDNA copies in the genomes of SZ patients is increased; therefore, we expect that the concentration of cell-free rDNA in the plasma of the SZ patients also increases. This may be one of the explanations of the proinflammatory cytokine increase that is often observed in SZ. The major research question is what is the rDNA copy number in cfDNA (cf-rDNA CN) and its putative role in schizophrenia? Materials and Methods. We determined cfDNA concentration (RNase A/proteinase K/solvent extraction; fluorescent dye PicoGreen) and endonuclease activity (NA) of blood plasma (radial diffusion method) in the untreated male SZ group (N = 100) and in the male healthy control group (HC) (N = 96). Blood leukocyte DNA and cfDNA rDNA CN were determined with nonradioactive quantitative hybridization techniques. Plasma concentration of cf-rDNA was calculated. RESULTS: In the subjects from the SZ group, the mean cfDNA plasma concentration was twofold higher and NA of the plasma was fourfold higher than those in the healthy controls. rDNA CN in the blood leukocyte genome and in the cfDNA samples in the SZ group was significantly higher than that in the HC group. cf-rDNA concentration was threefold higher in the SZ group. CONCLUSION: Despite the abnormally high endonuclease activity in the blood plasma of SZ patients, the circulating cfDNA concentration is increased. Fragments of cf-rDNA accumulate in the blood plasma of SZ patients. Potentially, SZ patients' cfDNA should be a strong stimulating factor for the TLR9-MyD88-NF-κB signaling pathway.

Oxidized Cell-Free DNA Role in the Antioxidant Defense Mechanisms under Stress.

The present study focuses on the investigation of the oxidized cell-free DNA (cfDNA) properties in several experimental models, including cultured cerebellum cells, peripheral blood lymphocytes (PBL), plasma, and hippocampus under an acute and chronic unpredictable stress model in rats. Firstly, our study shows that Spectrum Green fluorescence-labeled oxidized cfDNA fragments were transferred into the cytoplasm of 80% of the cerebellum culture cells; meanwhile, the nonoxidized cfDNA fragments do not pass into the cells. Oxidized cfDNA stimulates the antioxidant mechanisms and induction of transcription factor NRF2 expression, followed by an activation of NRF2 signaling pathway genes-rise of Nrf2 and Hmox1 gene expression and consequently NRF2 protein synthesis. Secondly, we showed that stress increases plasma cfDNA concentration in rats corresponding with the duration of the stress exposure. At the same time, our study did not reveal any significant changes of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) level in PBL of rats under acute or chronic stress, probably due to the significantly increased Nrf2 expression, that we found in such conditions. 8-oxodG is one of the most reliable markers of DNA oxidation. We also found an increased level of 8-oxodG in the hippocampal homogenates and hippocampal dentate gyrus in rats subjected to acute and chronic stress. Taken together, our data shows that oxidized cfDNA may play a significant role in systemic and neuronal physiological mechanisms of stress and adaptation.

Reinforced Hybrid Collagen Sponges for Tissue Engineering.

We created an anisotropic material based on collagen sponge and reactive polylactide structured by laser photopolymerization. The combination of collagen with reactive polylactide improves the resistance of the formed matrices to biodegradation in comparison with collagen sponge, while the existence of sites with different mechanical characteristics and cell affinity on the matrix provides directed cell growth during their culturing. It was shown that reinforcement of the collagen sponges 7-fold increased the mean Young's modulus for the hybrid matrix without affecting its cytotoxicity. The developed matrix provides cell adhesion and proliferation along reinforcement lines and can be used for fabrication of tissue engineering constructs.

ROS-Induced DNA Damage Associates with Abundance of Mitochondrial DNA in White Blood Cells of the Untreated Schizophrenic Patients.

OBJECTIVE: The aim of this study was (1) to examine the leukocyte mtDNA copy number (CN) in unmedicated (SZ (m-)) and medicated (SZ (m+)) male patients with paranoid schizophrenia (SZ) in comparison with the healthy male controls (HC) and (2) to compare the leukocyte mtDNA CN with the content of an oxidation marker 8-oxodG in lymphocytes of the SZ (m-) patients. METHODS: We evaluated leukocyte mtDNA CN of 110 subjects with SZ in comparison with 60 male HC by the method qPCR (ratio mtDNA/nDNA (gene B2M) was detected). SZ patients were divided into two subgroups. The patients of the subgroups SZ (m+) (N = 55) were treated with standard antipsychotic medications in the hospital. The patients of the subgroup SZ (m-) (N = 55) were not treated before venous blood was sampled. To evaluate oxidative DNA damage, we quantified the levels of 8-oxodG in lymphocytes (flow cytometry) of SZ (m-) patients (N = 55) and HC (N = 30). RESULTS: The leukocyte mtDNA CN showed no significant difference in SZ (m+) patients and HC. The mtDNA CN in the unmedicated subgroup SZ (m-) was significantly higher than that in the SZ (m+) subgroup or in HC group. The level of 8-oxodG in the subgroup SZ (m-) was significantly higher than that in HC group. CONCLUSION: The leukocytes of the unmedicated SZ male patients with acute psychosis contain more mtDNA than the leukocytes of the male SZ patients treated with antipsychotic medications or the healthy controls. MtDNA content positively correlates with the level of 8-oxodG in the unmedicated SZ patients.

Abundance of ribosomal RNA gene copies in the genomes of schizophrenia patients.

OBJECTIVE: The ribosome is a critical component of the translation machinery. The key component of ribosomes is ribosomal RNA (rRNA). Dysregulation of rRNA biogenesis has been implicated in some human diseases. One of the factors affecting rRNA biogenesis is the ribosomal RNA genes (rDNA) copy number in the genome. The aim of this study was to examine the rDNA copy number (CN) variation in the genomes of patients with schizophrenia (SZ) compared to healthy controls (HC). METHODS: We evaluated rDNA CN in leukocytes of 179 subjects with SZ (108 male/71 female) in comparison with 122 HC (60 male/62 female) using two techniques: qPCR and nonradioactive quantitative hybridization (NQH), which is based on the use of biotinylated rDNA probes. RESULTS: rDNA CN (NQH) and rDNA CN (qPCR) was higher in SZ patients than in controls (median 542 vs 384, p=10-25 and median 498 vs 370, p=10-12). NQH was experimentally proved to be less sensitive to severe DNA damage than qPCR. The more DNA damage, the higher the ratio R=CN (NQH)/CN (qPCR). 15% of the SZ patients had significantly higher rDNA damage degree than the HC. CONCLUSION: Genomes of some SZ patients contain more ribosomal genes than those of HC. The elevated ribosomal genes copy number in human genome can be one of the genetic factors of schizophrenia development. This hypothesis requires further experimental studies to be corroborated or disproved.

Low-Dose Ionizing Radiation Affects Mesenchymal Stem Cells via Extracellular Oxidized Cell-Free DNA: A Possible Mediator of Bystander Effect and Adaptive Response.

We have hypothesized that the adaptive response to low doses of ionizing radiation (IR) is mediated by oxidized cell-free DNA (cfDNA) fragments. Here, we summarize our experimental evidence for this model. Studies involving measurements of ROS, expression of the NOX (superoxide radical production), induction of apoptosis and DNA double-strand breaks, antiapoptotic gene expression and cell cycle inhibition confirm this hypothesis. We have demonstrated that treatment of mesenchymal stem cells (MSCs) with low doses of IR (10 cGy) leads to cell death of part of cell population and release of oxidized cfDNA. cfDNA has the ability to penetrate into the cytoplasm of other cells. Oxidized cfDNA, like low doses of IR, induces oxidative stress, ROS production, ROS-induced oxidative modifications of nuclear DNA, DNA breaks, arrest of the cell cycle, activation of DNA reparation and antioxidant response, and inhibition of apoptosis. The MSCs pretreated with low dose of irradiation or oxidized cfDNA were equally effective in induction of adaptive response to challenge further dose of radiation. Our studies suggest that oxidized cfDNA is a signaling molecule in the stress signaling that mediates radiation-induced bystander effects and that it is an important component of the development of radioadaptive responses to low doses of IR.

Quantification of cell-free DNA in blood plasma and DNA damage degree in lymphocytes to evaluate dysregulation of apoptosis in schizophrenia patients.

Oxidative DNA damage has been proposed as one of the causes of schizophrenia (SZ), and post mortem data indicate a dysregulation of apoptosis in SZ patients. To evaluate apoptosis in vivo we quantified the concentration of plasma cell-free DNA (cfDNA index, determined using fluorescence), the levels of 8-oxodG in cfDNA (immunoassay) and lymphocytes (FL1-8-oxodG index, flow cytometry) of male patients with acute psychotic disorders: paranoid SZ (total N = 58), schizophreniform (N = 11) and alcohol-induced (N = 14) psychotic disorder, and 30 healthy males. CfDNA in SZ (N = 58) does not change compared with controls. In SZ patients. Elevated levels of 8-oxodG were found in cfDNA (N = 58) and lymphocytes (n = 45). The main sources of cfDNA are dying cells with oxidized DNA. Thus, the cfDNA/FL1-8-oxodG ratio shows the level of apoptosis in damaged cells. Two subgroups were identified among the SZ patients (n = 45). For SZ-1 (31%) and SZ-2 (69%) median values of cfDNA/FL1-8-oxodG index are related as 1:6 (p < 0.0000001). For the patients with other psychotic disorders and healthy controls, cfDNA/FL1-8-oxodG values were within the range of the values in SZ-2. Thus, apoptosis is impaired in approximately one-third of SZ patients. This leads to an increase in the number of cells with damaged DNA in the patient's body tissues and may be a contributing cause of acute psychotic disorder.

Functionalized Fullerene Increases NF-κB Activity and Blocks Genotoxic Effect of Oxidative Stress in Serum-Starving Human Embryo Lung Diploid Fibroblasts.

The influence of a water-soluble [60] fullerene derivative containing five residues of 3-phenylpropionic acid and a chlorine addend appended to the carbon cage (F-828) on serum-starving human embryo lung diploid fibroblasts (HELFs) was studied. Serum deprivation evokes oxidative stress in HELFs. Cultivation of serum-starving HELFs in the presence of 0.1-1 µM F-828 significantly decreases the level of free radicals, inhibits autophagy, and represses expression of NOX4 and NRF2 proteins. The activity of NF-κB substantially grows up in contrast to the suppressed NRF2 activity. In the presence of 0.2-0.25 µM F-828, the DSB rate and apoptosis level dramatically decrease. The maximum increase of proliferative activity of the HELFs and maximum activity of NF-κB are observed at these concentration values. Conclusion. Under the conditions of oxidative stress evoked by serum deprivation the water-soluble fullerene derivative F-828 used in concentrations of 0.1 to 1 µM strongly stimulates the NF-κB activity and represses the NRF2 activity in HELFs.

[The maternal effect in infantile autism: elevated DNA damage degree in patients and their mothers]. / Materinskii éffekt pri detskom autizme: povyshennyi uroven' povrezhdenii DNK u patsientov i ikh materei.

Infantile autism is a common disorder of mental development, which is characterized by impairments in the communicative, cognitive and speech spheres and obsessional stereotyped behaviour. Although in most cases, pathogenic factors remain unclear, infantile autism has a significant hereditary component, however, its etiology is also under the influence of environmental factors, including the condition of the mother's body during pregnancy ("maternal effect"). Oxidative stress is assumed to play a key role in the pathogenesis of infantile autism. It is known that oxidative stress has a prominent genotoxic effect, which is realized through inducing single and double strand breaks of the nuclear DNA. We evaluated the degree of DNA damage in patients with infantile autism and their mothers using DNA comet assay. The comet tail moment and DNA per cent ratio in the tail were assessed for each individual. The two parameters appeared to be strongly correlated (r=0.90). Mean and median values of both parameters were considerably higher in the sample of autistic children, than in age-matching healthy controls. Interestingly, these parameters were also elevated in healthy mothers of autistic children, with no difference from the values in the group of autistic children. The control group of healthy women of reproductive age, who had no children with autism, differed by the DNA comet tail moment from the group of mothers of autistic children, but did not differ significantly from the control group of healthy children. The results suggest that there are genotoxic factors in mentally healthy mothers of autistic children, which can determine the pathological process in the foeti via environmental "maternal effect" during gestation.

[Enrichment of extracellular DNA from the cultivation medium of human peripheral blood mononuclears with genomic CpG rich fragments results in increased cell production of IL-6 and TNF-a via activation of the NF-kB signaling pathway]. / Obogashchenie vnekletochnoi DNK sredy kul'tivirovaniia mononuklearov perifericheskoi krovi cheloveka CpG-bogatymi fragmentami genoma privodit k uvelicheniiu produktsii kletkami IL-6 i TNF-a putem aktivatsii signal'nogo puti NF-kB.

Previously, it was found that blood plasma extracellular DNA (ecDNA) of patients with rheumatoid arthritis (RA) is enriched with CpG-rich genomic DNA fragments, which contain TLR9 ligands (Veiko et al., 2006). In this study we have demonstrated that ecDNA of a RA patient and model fragments added to a cultivation medium of peripheral blood mononuclear cells (PBMC) of healthy donors stimulate expression of genes for the TLR9-MyD88-NF-kB signaling pathway; this leads to a significant increase in concentrations of the proinflammatory cytokines IL-6 and TNF-a in the cultivation medium. Human genomic DNA non-enriched with the CpG sequences did not stimulate IL-6 and TNF-a synthesis in PBMC. A scheme explaining the potential role ecDNA in the induction and maintenance of increased levels of the proinflammatory cytokines under conditions damaging the human cells has been proposed.

Toxic and DNA damaging effects of a functionalized fullerene in human embryonic lung fibroblasts.

Water-soluble fullerenes have been studied as potential nanovectors and therapeutic agents, but their possible toxicity is of concern. We have studied the effects of F-828, a soluble fullerene [C60] derivative, on diploid human embryonic lung fibroblasts (HELFs) in vitro. F-828 causes complex time-dependent changes in ROS levels. Inhibition of Nox4 activity by plumbagin blocks F-828-dependent ROS elevation. F-828 induces DNA breaks, as measured by the comet assay and γH2AX expression, and the activities of the transcription factors NF-kB and p53 increase. F-828 concentrations>25µM are cytotoxic; cell death occurs by necrosis. Expression levels of TGF-ß, RHOA, RHOC, ROCK1, and SMAD2 increase following exposure to F-828. Our results raise the possibility that fullerene F-828 may induce pulmonary fibrosis in vivo.

RSH ANTIOXIDANTS INCREASE INTRACELLULAR ROS LEVEL AND PROLIFERATION OF SP2/0 MYELOMA CELLS IN SERUM-FREE MEDIUM.

At present research we have demonstrated the link between introducing of low molecular weight RSH-antioxidants (N-acetylcysteine, glutathione) into serum-free medium composition, reactive oxygen species (ROS) generation and mouse myeloma SP2/0-SF cells proliferative activity. The presence of these compounds in the medium changed the pattern of ROS-activity in cells by concentration-dependent manner, and affected their proliferative characteristics. The optimal value of the proliferative activity was related to 0.2 mM for both thyols and not depended from the thyol-compound nature. Further increasing up from the found concentration optimum lead to growth inhibition with different expression for N-acetylcysteine and glutathione.

Oxidized extracellular DNA suppresses nitric oxide production by endothelial NO synthase (eNOS) in human endothelial cells (HUVEC).

Circulating DNA from patients with cardiovascular diseases reduce the synthesis of NO in endothelial cells, which is probably related to oxidative modification of DNA. To test this hypothesis, HUVEC cells were cultured in the presence of DNA containing ~1 (nonoxidized DNA), 700, or 2100 8-oxodG/10(6) nucleosides. Nonoxidized DNA stimulated the synthesis of NO, which was associated with an increase in the expression of endothelial NO synthase. Oxidized NO decreased the amount of mRNA and protein for endothelial NO synthase, but increased the relative content of its low active form. These changes were accompanied by reduction of NO production. These findings suggest that oxidative modification of circulating extracellular DNA contributes to endothelial dysfunction manifested in suppression of NO production.

Enhanced expression of iNOS in human endothelial cells during long-term culturing with extracellular DNA fragments.

NO synthesis by endothelial cells plays an important role in normal function of the cardiovascular system. This work was designed to evaluate the role of variations in properties of extracellular DNA in the regulation of NO synthesis. We studied the effect of four DNA samples with various base sequences (50 ng/ml) on functional activity of endothelial cells HUVEC during 24-h culturing. Human DNA fragments with high content of CG repeats increased intracellular content of NO and its metabolites (nitrites and nitrates) and accelerated oxidation of nitrites to nitrates. Changes in the content of NO metabolites after 24-h culturing was shown to depend on the expression of gene for inducible, but not for endothelial NO synthase. Increased expression of inducible NO synthase positively correlated with an increase in the content of mRNA for the adapter protein MyD88, but did not depend on TLR9 gene expression that encodes protein receptor for CG-DNA recognition. The intracellular concentration of MyD88 mRNA did not depend on the content of TLR9 mRNA. The existence of a variety of DNA-binding receptors apart from TLR9 receptor on the surface of endothelial cells was hypothesized. Activation of these receptors by extracellular DNA fragments stimulates expression of the adapter protein MyD88.

Extracellular DNA affects NO content in human endothelial cells.

Fragments of extracellular DNA are permanently released into the blood flow due to cell apoptosis and possible de novo DNA synthesis. To find out whether extracellular DNA can affect the synthesis of nitric oxide (NO), one of key vascular tone regulators, we studied in vitro effects of three artificial DNA probes with different sequences and 10 samples of extracellular DNA (obtained from healthy people and patients with hypertension and atherosclerosis) on NO synthesis in endothelial cell culture (HUVEC). For detection of NO in live cells and culture medium, we used a NO-specific agent CuFL penetrating into the cells and forming a fluorescent product FL-NO upon interaction with NO. Human genome DNA fragments affected the content of NO in endothelial cells; this effect depended on both the base sequence and concentration of DNA fragments. Addition of artificial DNA and extracellular DNA from healthy people into the cell culture in a low concentration (5 ng/ml) increased the detected NO concentration by 4-fold at most. Cytosine-guanine (CG)-rich fragment of the transcribed sequence of ribosomal repeat was the most powerful NO-inductor. The effect of DNA fragments on NO synthesis was comparable with that of low doses of oxidizing agents, H(2)O(2) and 17ß-estradiol. Extracellular DNA samples obtained from patients with hypertension and atherosclerosis decreased NO content in cells and medium by 1.3-28 times compared to the control; the effect correlated with the content of CG-rich sequences.

Peripheral blood serum from healthy donors contains antibodies against the fragment of transcribed region of ribosomal repeat.

Enzyme immunoassay showed that blood serum from healthy donors contains specific high-affinity antibodies (apparent association constant > or = 5 x 10(9) M(-1)) against a fragment of transcribed region of ribosomal DNA repeat of human serum, which are present in a free form or are bound to extracellular DNA. Preheating of the serum at 55 degrees C and high ionic strength (1.5 M NaCl) had no effect on the interaction of antibodies with this fragment. Competitive binding assay showed that these antibodies recognize DNA epitopes, which differ from the epitopes recognized by most anti-DNA antibodies in systemic lupus erythematosus.

Stimulatory effect of fragments from transcribed region of ribosomal repeat on human peripheral blood lymphocytes.

Fragments from the transcribed region of the ribosomal repeat include considerable amounts of unmethylated CpG DNA motifs. These motifs activate immune cells via the interaction with Toll receptors. In vitro experiments confirmed the stimulatory effect of transcribed region of ribosomal repeat on human lymphocytes. Culturing of lymphocytes in a medium containing 2-20,000 ng/ml fragments from transcribed region of ribosomal repeat was accompanied by structural changes in the nucleus in a considerable number of cells. These changes manifested in translocation of pericentromeric heterochromatin from the membrane to the center of the nucleus and activation of the nucleolus and were accompanied by a significant increase in interleukin-6 production and slight stimulation of tumor necrosis factor-a synthesis. The transcribed region of the ribosomal repeat and E. coli DNA had various effects on quantitative parameters of lymphocytes. Our results suggest the existence of mechanisms of stimulation not mediated by the interaction of CpG DNA motifs with Toll receptors.